The present invention is in the field of biopesticides. More particularly, this invention relates to a novel strain of Streptomyces with insecticidal activity and methods of use thereof.
Natural products are substances produced by microbes, plants, and other organisms. Microbial natural products offer an abundant source of chemical diversity. There is a long history of utilizing these natural products for pharmaceutical purposes. Despite the emphasis on natural products for human therapeutics, there are only a few natural product insecticides for agricultural use. The most successful microbial natural product insecticides are the Bacillus thuringiensis toxins, avermectin and the spinosyns. Bacillus thuringiensis (Bt) bacteria produce cytoplasmic protein crystals (xcex4-endotoxins) during sporulation which are the most important factor in insect pathogenesis (see, Ellar, D. J. (1997) xe2x80x9cThe structure and function of Bacillus thuringiensis xcex4-endotoxins and prospects for biopesticide improvement,xe2x80x9d In: Microbial Insecticides: Novelty or Necessity? The British Crop Protection Council Symposium Proceedings No. 68, Coventry, UK). The xcex4-endotoxins have been used in both spray preparations as well as xe2x80x98systemicxe2x80x99 biopesticides through the introduction of the endotoxin genes into transgenic plants. The growth of the market for the spray preparations of Bt is estimated to be around 10% for 1997 through 2000, which will result in a market of $100-$130 million by 2000 (see, Lisansky, S. (1997) xe2x80x9cMicrobial biopesticidesxe2x80x9d In: Microbial Insecticides: Novelty or Necessity? The British Crop Protection Council Symposium Proceedings No. 68, Coventry, UK.). The main Bt transgenic crops are maize and cotton. The transgenic Bt maize market in the U.S. has grown from only 1.4% of the planted acreage in 1996 to 19.1% in 1998. The growth in Bt cotton has not been as drastic, increasing from 14.6% of the planted acreage in 1996 to 16.8% in 1998. The entire market is currently directed against lepidopteran pests. In 1999, the market for pesticides used against caterpillars in the US exceeded 400 million dollars US.
The avermectins are produced by Streptomyces avermitilis during fermentation. Abamectin, one of the naturally occurring macrocyclic lactones, shows activity against mites, pear psylla and diamond back moth. Emamectin, a semi-synthetic analog of abamectin, shows activity against lepidopteran larvae. In invertebrates the avermectins induce the opening of a pre-synaptic chloride ion channel (not GABA-activated), leading to efflux of chloride ions, depolarization of the nerve terminal, and hence, to neurotransmitter release. See, Turner, M. J. and Schaeffer, J. M. (1989) xe2x80x9cMode of action of Ivermectin,xe2x80x9d In: Ivermectin and Abamectin, W. C. Campbell (Ed.) Springer-Verlag, N.Y. The use of avermectins in insect control had an estimated world market value of $80-$120 million in 1998.
The spinosyns are a new class of fermentation-derived tetracyclic-macrolides produced by the actinomycete Saccharopolyspora spinosa. Spinosyns A and D, the principal components of the spinosad insecticide Tracer(copyright), show activity against lepidopteran pests and mosquitoes. See, Sparks, T. C. et al. (1999) xe2x80x9cFermentation-derived insecticide control agents: the spinosynsxe2x80x9d In: Biopesticides Use and Delivery, Hall, F. R. et al., eds. Humana Press, Totowa, N.J., pp.155-170. Its mode of action is unique, with a primary site of attack on the nicotine acetylcholine receptor and a secondary attack, possibly on or at GABA receptors. See, Salgado, V. L. (1997) xe2x80x9cThe modes of action of spinosad and other insect control productsxe2x80x9d Down to Earth 52:35-43.
Streptomyces are a recognized source of insecticidal natural products. In addition to the avermectins and spinosyns, cholesterol oxidase (Purcell, J. P. et al. (1993) Biochem. Biophys. Res. Commun. 196:1406-1413), allosamidin (Sakuda, S. (1986) Tetrahedron Lett. 27:2475-2478), valinomycin (Heisey, R. (1988) J. Agric. Food Chem. 36:1283-1286), pyrrolizine derivatives (Jizba, J. et al. (1992) Folia-Microbiologica 37:461-462), respirantin (Urushibata, I. et al. (1993) J. Antibiotics 46:701-703), prasinons (Box, S. J. et al. (1973) Appl. Microbiol. 26:699-704), piercidin (Takahashi, N. et al. (1968) Agr. Biol. Chem. 32:1115-1122) griseulin (Nair, M. G. et al. (1993) J. Antibiotics 46:1762-1763), martinomycin (Carter, G. T. et al. (1994) J. Antibiotics 47:1549-1553), faeriefungin (Nair, M. G. et al. (1989) J. Nat. Prod. 52:797-809), indanomycin (Zhang, D. et al. (1997) J. Antibiotics 50:617-620), cyclophostin (Kurokawa, T. et al. (1993) J. Antibiotics 46:1315-1318), manumycin (Zeeck, A. et al. (1987) J. Antibiotics 40:1530-1540), ichthyomycin (Zizka, Z. et al. (1991) Cytobios 65:31-38), virginiamycin (Prikrylova, V. et al. (1992) Folia Microbiol. 37:386-388), suidatestrin (Knuessel, I., et al. (1998) Comp. Biochem. Phys. B. 120B:639-646), gualamycin (Tsuchiya, K. J. (1995) J. Antibiotics 48:626-629), and other insecticidal natural products have been reported from Streptomyces strains.
Streptomyces galbus is recognized for its production of the potent anti-botrytis macrolides, galbonolide A and galbonolide B. (Paul A. K. and Banerjee, A. K. (1983) Folia Microbiol. 28:386-396; Sigmund, J. M. and Hirsch, C. F. (1998) J. Antibiotics 51:829-836; Achenbach, H., DE Patent No. 86-3632168; and Zaehner, H., DE Patent No. 3632168). Nakayama et al. (1987) Agric. Biol. Chem. 51:853-860, reported the discovery of rustmicin, a potent inhibitor of wheat stem rust, Puccinia gramnis. Later, galbonolide A was shown to be the same molecule as rustmicin. Achenbach et al. (1988) Annals N.Y. Acad. Sci. 544:128-140, reported galbonolide A to be much more active than galbonolide B against a number of endomycetous yeasts and a large number of Deuteromycetes such as Candida albicans and Botrytis cinerea. No evidence for ionophoric activity, membrane destabilization, interference with DNA or RNA biosynthesis, or inhibition of chitin biosynthesis could be attributed to these macrolides. Recently Mandala et al. (1998)J. Biol. Chem. 24:14942-14949) and Harris et al. (1998) J. Antibiotics 51:837-844; and Harris, G., et al. Patent No. GB 2324300) demonstrated that rustmicin inhibits inositol phosphoceramide synthetase, the first fungal-specific-enzyme in sphingolipid biosynthesis. We now document the potent insecticidal properties of a novel strain of S. galbus, with activity against a number of agriculturally relevant Lepidoptera.
The present invention provides a novel compound, Streptomyces galbus, NRRL Accession No. 30232, and mutants thereof which retain the same activity, for use as an insecticide against Lepidoptera. The invention encompasses the use of supernatants and metabolites from the strain for use as an insecticide. The invention also includes methods of treating plants or fruit to control Lepidoptera infestations using the claimed strain, either alone, or in combination with other chemical or biological pesticides. Further provided are methods to ferment the claimed strain to increase its bioactivity as an insecticide.
Deposit of Microorganisms
A strain of Streptomyces galbus was deposited on Dec. 10, 1999 according to the Budapest Treaty in the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 University Street, Peoria, Illinois, 61604, USA. The Accession number is NRRL 30232.
The strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this application. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Definitions
As used herein, certain terms may have the following defined meanings.
The singular form xe2x80x9ca,xe2x80x9d xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d include plural references unless the context clearly dictates otherwise. For example, the term xe2x80x9ca cellxe2x80x9d includes a plurality of cells, including mixtures thereof.
The term xe2x80x9ccomprisingxe2x80x9d is intended to mean that the compositions and methods include the recited elements, but not excluding others. xe2x80x9cConsisting essentially ofxe2x80x9d when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and agriculturally acceptable carriers. xe2x80x9cConsisting ofxe2x80x9d shall mean excluding more than trace elements of other ingredients and substantial method steps for applying the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
As used herein, xe2x80x9cbiological controlxe2x80x9d is defined as control of a pathogen or insect by the use of a second organism. Known mechanisms of biological control include enteric bacteria that control root rot by out-competing fungi for space on the surface of roots, or the application of Bacillus thuringiensis to control insect pests. Bacterial toxins, such as antibiotics, have been used to control pathogens and insects. The toxin can be isolated and applied directly to the plant or the bacterial species may be administered so it produces the toxin in situ.
The term xe2x80x9cfungusxe2x80x9d or xe2x80x9cfungixe2x80x9d includes a wide variety of nucleated spore-bearing organisms that are devoid of chlorophyll. Examples of fungi include yeasts, molds, mildews, rusts, and mushrooms.
The term xe2x80x9cbacteriaxe2x80x9d includes any prokaryotic organism that does not have a distinct nucleus.
xe2x80x9cFungicidalxe2x80x9d or xe2x80x9canti-fungalxe2x80x9d means the ability of a substance to increase mortality or inhibit the growth rate of fungi.
xe2x80x9cAntibioticxe2x80x9d includes any substance that is able to kill or inhibit the growth of another living organism, including, but not limited to other microorganisms. Antibiotics may be produced by a microorganism or by a synthetic process or semi-synthetic process.
The term xe2x80x9cmutantxe2x80x9d refers to a variant of the parental strain as well as methods for obtaining a mutant or variant in which the pesticidal activity is greater than that expressed by the parental strain. The xe2x80x9cparent strainxe2x80x9d is defined herein as the original Streptomyces strain before mutagenesis. To obtain such mutants the parental strain may be treated with a chemical such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine, ethylmethanesulfone, or by irradiation using gamma, x-ray, or UV-irradiation, or by other means well known to those practiced in the art.
A xe2x80x9cvariantxe2x80x9d is a strain having all the identifying characteristics of NRRL Accession No. 30232 and can be identified as having a genome that hybridizes under conditions of high stringency to the genome of NRRL Accession No. B-30232. xe2x80x9cHybridizationxe2x80x9d refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. Hybridization reactions can be performed under conditions of different xe2x80x9cstringency.xe2x80x9d In general, a low stringency hybridization reaction is carried out at about 40xc2x0 C. in 10xc3x97SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50xc2x0 C. in 6xc3x97SSC, and a high stringency hybridization reaction is generally performed at about 60xc2x0 C. in 1xc3x97SSC.
A variant of NRRL Accession No. 30232 may also be defined as a strain having a genomic sequence that is greater than 85%, more preferably greater than 90% or more preferably greater than 95% sequence identity to the genome of NRRL Accession No.
B-30232. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of xe2x80x9csequence identityxe2x80x9d to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.
The term xe2x80x9cculturingxe2x80x9d refers to the propagation of organisms on or in media of various kinds.
xe2x80x9cWhole broth culturexe2x80x9d refers to a liquid culture containing both cells and media.
xe2x80x9cSupernatantxe2x80x9d refers to the liquid broth remaining when cells grown in broth are removed by centrifugation, filtration, sedimentation, or other means well known in the art.
An xe2x80x9ceffective amountxe2x80x9d is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. In terms of treatment and protection, an xe2x80x9ceffective amountxe2x80x9d is that amount sufficient to reduce, retard, or eliminate an insect""s ability to feed, grow, and reproduce in the pre-adult or adult stage of development.
As used herein, the term xe2x80x9cinsectxe2x80x9d includes all organisms in the class Insecta.
As used herein the term xe2x80x9cLepidopteraxe2x80x9d refers to the insect order characterized by a larval stage taking the form of a caterpillar or worm and an adult stage taking the form of a moth or a butterfly.
A xe2x80x9cpre-adultxe2x80x9d insect refers to any form of an organism prior to the adult stage, including, for example, eggs, larvae, and nymphs.
xe2x80x9cInsecticidalxe2x80x9d refers to the ability of a substance to increase mortality or inhibit the growth rate of insects.
xe2x80x9cPesticidalxe2x80x9d refers to the ability of a substance to increase mortality or inhibit the growth rate of insects, nematodes and mites.
xe2x80x9cPositive controlxe2x80x9d means a compound known to have pesticidal activity. xe2x80x9cPositive controlsxe2x80x9d include, but are not limited to commercially available chemical pesticides.
The term xe2x80x9cnegative controlxe2x80x9d means a compound known not to have pesticidal activity. Examples of negative controls are water, media blanks, or low concentrations of solvent such as acetone, ethanol, or ethyl acetate.
Media blanks are composed of sterilized fermentation broth in the absence of a fermenting microorganism.
The term xe2x80x9csolventxe2x80x9d includes any liquid that holds another substance in solution.
xe2x80x9cSolvent extractablexe2x80x9d refers to any compound that dissolves in a solvent and which then may be isolated from the solvent. Examples of solvents include, but are not limited to, organic solvents like ethyl acetate (EtOAc), acetone, ethanol (EtOH), acetonitriles, methanol (MeOH), butanol (BuOH), or dimethylsulfoxide (DMSO).
The term xe2x80x9cmetabolitexe2x80x9d refers to any compound, substance or by product of the fermentation of a microorganism that has biological activity.
A xe2x80x9ccompositionxe2x80x9d is intended to mean a combination of active agent and another compound, carrier or composition, inert (for example, a detectable agent or label or liquid carrier) or active, such as an adjuvant.
This invention provides a biologically pure culture of a strain having all the identifying characteristics of Streptomyces galbus, NRRL Accession No. 30232, and its variants and mutants, as described above, that have insecticidal activity. In one embodiment, the invention is the strain designated NRRL Accession No. 30232.
This invention provides a process for isolating a metabolite from the biologically pure cultures of strains described herein as well as the metabolites isolated thereby. The metabolite is characterized as being solvent extractable and having a molecular weight of less than 10,000 Daltons.
Further provided is a process for purifying biologically active supernatants from the biologically pure cultures identified herein and the supernatants isolated thereby. The isolated supernatants have insecticidal activity similar to that of a Streptomyces galbus, NRRL Accession No. 30232, or mutants and variants thereof.
Also provided by this invention are compositions comprising at least one of the biologically pure cultures, supernatants or the isolated metabolite described above and a carrier. In another aspect, the composition further contains at least one chemical or biological pesticide. The compositions are formulated as any one or more of a wettable powder, a granule, an aqueous suspension, and emulsifiable concentrate and a microencapsulated formulation.
In order to achieve good dispersion and adhesion of compositions within the present invention, it may be advantageous to formulate the whole broth culture, supernatant, fraction and/or metabolite/antibiotic with components that aid dispersion and adhesion. Accordingly, suitable formulations will be known to those skilled in the art (wettable powders, granules and the like, or can be microencapsulated in a suitable medium and the like, liquids such as aqueous flowables and aqueous suspensions, and emulsifiable concentrates). Other suitable formulations will be known to those skilled in the art.
Any of the above noted strains, metabolites, fractions, supernatants and compositions containing these active ingredients, may be used to provide a method of treating or protecting plants, roots or fruit from insect infections. Insects include, but are not limited to an insect selected from the group consisting of Lepidoptera, Coleoptera and Diptera. In one aspect, the insect is Lepidoptera, e.g., Spodoptera exigua, Anticarsia gemmatalis, Plutella spp., Helicoverpa zea, Heliothis virescens, and Trichoplusia ni. 
Also provided by this invention is a method for producing a supernatant active as an insecticide by culturing the strains of this invention and isolating the supernatant. The supernatant produced by this method is also claimed herein.
The metabolite is isolated by reverse phase, solid phase extraction using a step gradient of methanol and water. The metabolite can be identified by its molecular weight (less than 10,000 Daltons).